Glycation represents the chain reactions starting from the non-enzymatic reaction between the amino moiety on peptides or proteins and the carbonyl moiety on reducing sugars (Maillard reaction; cf. Reference 1) and are divided roughly into the initial stage and the later stage. The initial stage comprises a reversible reaction depending on the concentration of sugars and the reaction time wherein the amino moiety and the carbonyl moiety are non-enzymatically reacted to form Schiff bases, followed by Amadori rearrangement to form Amadori compounds.
In the later stage, the Amadori compounds formed in the initial stage are irreversibly subjected to dehydration, condensation, cyclization, oxidation, fragmentation, polymerization, rearrangement, etc. to give finally protein modification products called “AGEs”. By auto-oxidation of sugars and the like, there are produced highly reactive dicarbonyl compounds such as 3-deoxiglucosone (hereinafter referred to as “3-DG”), glyoxal (hereinafter referred to as “GO”) and methylglyoxal (hereinafter referred to as “MGO”), which may be further reacted with proteins to form AGEs modified at the lysine or arginine residues of the proteins in many cases.
Under the oxidation stress conditions, sugars, lipids, amino acids, etc. present abundantly in living bodies are oxidized to highly reactive carbonyl compounds. The thus produced GO, MGO, arabinose, glycol aldehyde, etc. are served as precursors of AGEs. Dehydroascorbic acid, which is formed by oxidation of ascorbic acid, is also served as a precursor of AGEs. These precursors have a carbonyl group, which is reacted non-enzymatically reacted with the amino moiety on proteins to give Schiff's bases and then form AGEs (cf. Reference 2).
Under the oxidation stress condition, lipoperoxidation also proceeds to form various carbonyl compounds such as malondialdehyde, hydroxynonenal and acrolein (cf. Reference 3). These carbonyl compounds react with the amino moiety or the like on proteins to form protein modification products called ALEs such as malondialdehyde-modified lysine and hydroxynonenal modifier (cf. Reference 2).
In addition, amino acids such as serine and threonine are oxidized to form carbonyl compounds such as acrolein and GO, followed by conversion into protein modification products (cf. Reference 4). A large number of carbonyl compounds are formed by the oxidative pathway, but some carbonyl compounds such as 3-DG are formed through the non-oxidative pathway.
As the known pathways for production of AGEs, there are (i) the pathway of conversion of Schiff's bases or Amadori compounds into AGEs through 3-DG, (ii) the pathway of oxidative conversion of Schiff's bases into glycolaldehyde alkylimines, followed by conversion of the latter into AGEs via aldoamines, (iii) the pathway conversion of aldoamines into AGEs via glyoxal monoalkylimines, (iv) the pathway of conversion of Amadori comounds into MGO through 2,3-enediol, followed by conversion of said MGO into AGEs, and (v) others.
It has recently been revealed that carboxymethyl-lysine as one of the AGEs is produced from GO, which is formed by lipoxidation of unsaturated fatty acids. It is thus considered that the glycation/oxidation reaction and the lipoxidation reaction occur on the common basis.
As understood from the above, carbonyl compounds produced through the oxidative or non-oxidative pathway from sugars, lipids, amino acids and ascorbic acid, modify proteins non-enzymatically and finally give protein modification products such as AGEs and ALEs. In particular, the increased state of the protein modification reaction by carbonyl compounds formed via a plurality of reaction pathways is called the protein modification due to excessive carbonyl, i.e. “carbonyl stress”.
Known AGEs include pentosidine (cf. Reference 5), crossrine (cf. Reference 6), X1 (fluorolink), pyropyridine (cf. Reference 7), pyrarine (cf. Reference 8), carboxymethyl-lysine (cf. Reference 9), imidazolone compounds (cf. Reference 10), carboxyethyl-lysine (cf. Reference 11), MGO dimer (cf. Reference 12), GO dimer (cf. Reference 13), imidazolysine (cf. Reference 14), argupyrimidine (cf. Reference 15), etc.
AGEs receptors as heretofore cloned include RAGE (cf. Reference 16), macrophage scavenger receptor class A (cf. Reference 17), galectin 3 (cf. Reference 18), OST-48 and 80K-H (cf. Reference 17), etc.
It is reported that in the blood vessel tissue, RAGE (a cellular membrane penetration type protein belonging to the immunoglobulin superfamily) is bonded to AGEs, thereby active oxygen is generated in the cell to activate the p21ras/MAPK pathway (cf. Reference 19) so that the activation of the transcription factor NF-κB is induced to lead the expression of angiopathy associated factors such as VCAM-1 (cf. Reference 20). It is also reported that AGEs control the proliferation of endothelial cells in finer vessels via RAGE, control the proliferation of pericytes playing an important roll in homeostasis and produce a toxic effect (cf. Reference 21).
In addition, it is reported that AGEs act directly onto endothelial cells in finer vessels via RAGE to promote neoangiogenesis and inhibit the production of PG12 for thrombus tendency (cf. Reference 22). For further interests, enhancement of the substrate production in mesanginal cells, enhancement of the monocyte migration ability, release of inflammatory cytokines from macrophages, acceleration of the collagenase production in synovial cells, activation of osteoclasts, proliferation of vascular smooth muscle cells, acceleration of platelet aggregation, NO activity and its suppression of the smooth muscle relaxation are reported as the physiological activities of AGEs and ALEs (cf. Reference 23).
Diseases associated with AGEs include (i) nephropathy as a complication of diabetes (cf. Reference 24), nervous disorder (cf. Reference 25), retinopathy (cf. Reference 21) and cataract, (ii) arteriosclerosis (cf. Reference 26), (iii) dialysis amyloidosis as a complication of dialysis (cf. Reference 27) and peritoneal sclerosis in peritodialysis patients, (iv) Alzheimer's disease as a central neurological disease (cf. Reference 28), Pick's disease and Parkinson disease, (v) rheumatoid arthritis (cf. Reference 29), (vi) sunlight elastic fibrosis, (vii) aging, (viii) renal failure (cf. Reference 30), etc. In addition, it is reported that in case of diabetes, AGEs prevent the vasodilation derived from blood vessel endothelial cells (cf. Reference 31), and promote renal sclerosis (cf. Reference 32).
From the above, it is understood that protein modification products such as AGEs afford an adverse effect on living bodies directly or via receptors.
On the other hand, it is known that the blood concentration of AGEs are increased with the reduction of the renal function. The reduction of the renal function results in the accumulation of carbonyl compounds considered as having a molecular weight of not more than 5 kDa. In case of pentosidine or pyrarine, those can be present in a free form, but a large portion of them present in a binding form to serum albumin or the like (cf. Reference 33). In addition, it is reported that the blood level of pentosidine is strongly affected by the filtration function of glomeruli (cf. Reference 34).
In this way, a large portion of AGEs is eliminated from kidney, and their blood concentration is kept lower during good health. However, when the renal function is reduced, they act as a uremic toxin to produce a chronic bioactivities.
Dialysis therapy can remove AGEs in a free form but hardly remove those in a binding form to proteins or in an intramolecular bridging form (cf. Reference 35). Therefore, the accumulation of modified forms in living bodies is increased with the progression of renal failure. Further, in addition to the fundamental process where sugars are reacted in living bodies, AGEs in a free form supplied by diets as well as highly active intermediates such as 3-DG, GO and MGO formed from Amadori compounds and the like previously produced in living bodies react with proteins in succession to enhance the production of AGEs. Furthermore, the contact of blood to a dialysis membrane results, for instance, in activation of complements and leucocytes to enhance the generation of free radicals. Thus, dialysis therapy itself enhances oxidation and makes one of the causes for production of AGEs.
Accordingly, it is important in dialysis therapy to remove free form substances at an early stage of dialysis and suppress the generation of AGEs in a binding form as much as possible. Since it is difficult to remove AGEs in a binding form by dialysis therapy as stated above, development of a medicament which suppresses formation of protein modification products is highly desired for dialysis therapy.
Further, it is believed that not only reduction of renal function but also fall of anti-oxidation protective mechanism associated with renal failure is concerned with accumulation of protein modification products. In patients with renal failure, unbalance of such anti-oxidation abilities is suggested (cf. Reference 40) as the increase of oxidized glutathione against reducing glutathione in blood (cf. Reference 36), the reduction of activity of glutathione dependent enzymes, the decrease of preservation term renal failure plasma glutathione peroxidase (cf. Reference 37), the decrease of blood glutathione (cf. Reference 38) and the increase of activity of plasma superoxide dismutase against the decrease of selenium concentration in plasma (cf. Reference 39).
Furthermore, it is reported that in patients with chronic renal failure, remarkable amounts of highly reactive carbonyl compounds and AGEs are generally accumulated in blood and tissues regardless of hyperglycemia (cf. Reference 41). In renal failure, carbonyl compounds are placed under a state of high load (carbonyl stress) by non-enzymatic chemical reaction so that protein modification products are increased. This is considered to have been caused by modification of proteins with carbonyl compounds produced from sugars and lipids (cf. Reference 42).
Accordingly, suppression of the production of protein modification products caused by various factors may accomplish alleviation of tissue injury, and prevent or treat the conditions associated with protein modification products such as AGEs.
Dialysis for patients with chronic renal failure includes hemodialysis and peritoneal dialysis. In case of peritoneal dialysis, debris in blood is excreted into peritoneal dialysate through a peritoneal membrane. Peritoneal dialysate of high osmotic pressure, which contains glucose, icodextrin, amino acid or the like, is effective in collecting such highly reactive carbonyl compounds accumulated in blood of patients with renal failure as carbonyl compounds derived from carbohydrates (e.g. arabinose, GO, MGO, 3-DG), carbonyl compounds derived from ascorbic acid (e.g. dehydroascorbic acid) and carbonyl compounds derived from lipids (e.g. hydroxynonenal, malondialdehyde, acrolein) through a peritoneal membrane therein.
Further, it is known that highly reactive carbonyl compounds (e.g. 3-DG, 5-hydroxymethylfurfural, formaldehyde, acetaldehyde, GO, MGO, levulinic acid, furfural, arabinose) are formed in a peritoneal dialysate during the sterilization or storage of the peritoneal dialysate (cf. Reference 43).
Therefore, the concentration of carbonyl compounds in the peritoneal dialysate increases, and formation of protein modification enhances. As the result, the function of the peritoneal membrane is reduced, and thereby reduction of the water removing ability and progression to peritoneal sclerosis would be caused (cf. Reference 44).
In fact, it is demonstrated by the immunohistological study of endothelium and mesothelium that in patients with peritoneal dialysis, introduced glucose makes a carbonyl stress condition in the peritoneal cavity (cf. Reference 45).
In this way, it is presumed that in patients with dialysis, production of protein modification products by carbonyl compounds causes the morphological alteration of peritoneum and the reduction of the function (i.e. water removal function) resulting therefrom.
Taking into consideration the above facts and various morbid conditions such as renal failure in combination, it is believed that the accumulation of carbonyl compounds is one of the causes for enhancement of the AGEs production (cf. Reference 46). Thus, suppression of the AGEs production is considered as an effective measure for treatment of the conditions associated with AGEs.
A typical example of AGEs production inhibitors is aminoguanidine, which is considered to inhibit AGEs production by reaction with dicarbonyl compounds such as 3-DG generated from glucose, Schiff's bases or Amadori compounds to form thiazolines. Analysis using diabetes animal models confirmed that said compound is effective in delaying the progression of diabetic nephropathy (cf. Reference 47), retinopathy (cf. Reference 48) and cataract (cf. Reference 49).
Other examples are pyridoxamine derivatives (e.g. pyridorine). In case of OPB-9195 (i.e. (±)2-isopropylidene-hydrazon-4-oxo-thiazolydin-5-yl-acetanilide), the nitrogen atom in the hydrazine moiety is reacted with a carbonyl group to form a stable structure. Thus, it captures a reactive carbonyl group in a free form or a binding form to protein (cf. Reference 50) and therefore can prevent the production of not only AGEs but also ALEs in vitro. Since biguanide compounds such as metformin or buformin can also capture carbonyl compounds (cf. Reference 51), they have a possibility of being used as AGEs forming inhibitors. Further, the use of AGEs inhibitors capable of cleaving the bridge as a characteristic of AGEs and the enzymes capable of degrading Amadori compounds (i.e. amadoriase) are proposed.
Study is also made on the possibility of prevention of the AGEs and/or ALEs formation by removal of carbonyl compounds. For removal of carbonyl compounds, there are available several enzymes and enzymatic pathways, of which examples are aldol reducing enzymes and aldehyde dehydrogenase and glyoxalase pathway (cf. Reference 52). Redox co-enzymes such as reducing glutathione (GSH) and NAD(P)H are important factors for activation of those pathways.
Lowering of these removing pathways leads to increasing of numerous carbonyl compounds at the same time. Carbonyl compounds such as MGO and GO react with the thiol group of GSH and, as the result, they are metabolized with an enzyme, i.e. glyoxalase. NAD(P)H activates the glutathione reducing enzyme and enhances the GSH level. Namely, it is believed that the removal system of carbonyl compounds is inhibited by lowering of GSH or NAD(P)H due to unbalance of the intracellular redox mechanism, which leads to accumulation of AGEs and ALEs. In case of diabetes, it is suggested that the polyol pathway is activated by hyperglycemia, NAD(P)H and GSH are reduced and the removal system of carbonyl compounds is lowered.
If reduction in the concentration of thiols such as GSH and NAD(P)H lowers the removal of carbonyl compounds, and thereby causes the production of AGEs or ALEs as stated above, there is a possibility that carbonyl compounds would be decreased by increasing the thiol level. For this purpose, the supplementation of thiol groups with GSH, cysteine, acetylcysteine, etc., the lowering of the GSH demand with vitamin E, ubiquinol, etc. and the inhibition of the polyol system with aldose reducing enzyme inhibitors are proposed. Trapping of carbonyl compounds by the use of aminoguanidine, pyridoxamine, hydrazine, biguanide compounds or SH-containing compounds is also proposed (cf. Patent Reference 1).
As stated above in detail, the inhibitions of production of AGEs and ALEs are the way for prevention and treatment of diseases associated with them.
Patent Reference 1: WO 00/10606
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